Articles
In Ovo Exposure to o,p´-DDE Affects Sexual Development But Not Sexual
Differentiation in Japanese Medaka (Oryzias latipes)
Diana M. Papoulias,1,2 Sergio A. Villalobos,3 John Meadows,1 Douglas B. Noltie,2 John P. Giesy,4 and
Donald E. Tillitt1
1U.S. Geological Survey, Columbia Environmental Research Center, Columbia, Missouri, USA; 2University of Missouri, Department of
Fisheries and Wildlife, Columbia, Missouri, USA; 3Syngenta, Inc., Greensboro, North Carolina, USA; 4Michigan State University,
East Lansing, Michigan, USA
Despite being banned in many countries, dichlorodiphenyltrichloroethane (DDT) and its metabolites dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD) continue to be found in fish tissues at concentrations of concern. Like o,p´-DDT, o,p´-DDE is
estrogenic and is believed to exert its effects through binding to the estrogen receptor. The limited
toxicologic data for o,p´-DDE suggest that it decreases fecundity and fertility of fishes. We conducted an egg injection study using the d-rR strain of medaka and environmentally relevant concentrations of o,p´-DDE to examine its effects on sexual differentiation and development. The
gonads of exposed fish showed no evidence of sex reversal or intersex. However, other gonad
abnormalities occurred in exposed individuals. Females exhibited few vitellogenic oocytes and
increased atresia. Male testes appeared morphologically normal but were very small.
Gonadosomatic index values for both sexes were lower for exposed fish. Our observations of
abnormal female and very small male gonads after in ovo o,p´-DDE exposure may be indicative of
effects on early endocrine processes important for normal ovarian and testicular development. Key
words: egg injection, medaka, o,p´-DDE, sexual development, sexual differentiation, xenoestrogen.
Environ Health Perspect 111:29–32 (2003). [Online 8 November 2002]
doi:10.1289/ehp.5540 available via http://dx.doi.org/
In ovo exposure of fish to the synthetic estrogen ethinyl estradiol can cause genetic males
to sexually differentiate as phenotypic females
(Papoulias et al. 1999). Presumably, the binding of estrogen to the estrogen receptor (ER)
initiates the little-understood process of establishing the female phenotype in teleost fishes.
Because many man-made chemicals bind to
the ER, the question arises of whether environmentally exposed male fish are at risk of
being feminized.
Although use of dichlorodiphenyltrichloroethane (DDT) is banned in much of the world,
the parent compound and its metabolites persist in the global environment, throughout the
food chain, and thus remain a concern (Brown
1997; Munn and Gruber 1997; Schmitt et al.
1999; Simonich and Hites 1995). DDT and its
metabolites are highly lipophilic and therefore
easily bioaccumulate in fish. Ungerer and
Thomas (1996) demonstrated that breeding
females will accumulate o,p´-DDT within the
triglyceride-rich oil globule of the oocyte. Here,
the o,p´-DDT remains sequestered until egg
fertilization, after which it becomes available to
the developing embryo. The oil globule is generally the last endogenous nutritional source
used by the sac fry before they switch to exogenous feeding (Heming and Buddington 1988).
Thus, for many species, the greatest exposure
to the developing embryo from hydrophobic
estrogenic chemicals occurs during sexual differentiation (i.e., shortly after hatch).
Despite the diminished environmental
presence of DDT over the last 30 years, DDE
Environmental Health Perspectives
(dichlorodiphenyldichloroethylene) metabolites are still commonly detected (Adeshina and
Todd 1991; Brown 1997; Hellou et al. 1995;
Schecter et al. 1997). DDE is a breakdown
product of DDT, a recognized endocrine system modulator. However, very few laboratory
studies of the effects of DDE have been published, and of these, most report on the
p,p´-DDE isomer. The p,p´-isomers are far
more common than the o,p´-isomers, because
technical-grade DDT consists of approximately 80% p,p´-DDT and 20% o,p´-DDT.
The p,p´-isomers are considered androgen
receptor antagonists and not xenoestrogens
(Kelce et al. 1995).
Although o,p´-DDT has long been recognized as estrogenic (Bitman et al. 1968;
Kupfer and Bulger 1976), relatively little is
known about its metabolite o,p´-DDE.
However, like o,p´-DDT, o,p´-DDE is
believed to work through the ER and to have
a similar ER agonist potency (Donohoe and
Curtis 1996), and exposure to o,p´-DDE has
been associated with decreased fecundity and
fertility and increased early oocyte atresia
(Hose et al. 1989). Recently, Wells and Van
Der Kraak (2000) showed that o,p´-DDE and
o,p´-DDT will also bind to the androgen
receptor in goldfish (Carassius auratus) but not
rainbow trout (Oncorhynchus mykiss) testes at
approximately half the affinity of p,p´-DDE.
o,p´-DDE has even been shown to bind to the
progesterone receptor in the eggshell gland
mucosa of egg-laying ducks and domestic fowl
and to the endometrium of the rabbit uterus
• VOLUME 111 | NUMBER 1 | January 2003
(Lundholm 1988). Furthermore, DDT can be
metabolized to DDE by the developing fish
embryo (Atchison and Johnson 1975).
The objective of this study was to investigate the effects of o,p´-DDE on sexual differentiation and development. We accomplished
this through injection of environmentally relevant concentrations of o,p´-DDE into earlystage embryos of medaka (Oryzias latipes).
Materials and Methods
Test organism. The d-rR strain of medaka was
a generous gift of Akihiro Shima (University
of Tokyo, Tokyo, Japan). Broodstock were
maintained at the Columbia Environmental
Research Center (CERC) in well water under
an 18:6 hr light:dark photoperiod at 27°C. In
this strain, either R (orange-red) or r (white) is
carried by the X or Y chromosome, respectively. White females (X r X r ) crossed with
orange-red males (X rY R) produce approximately equal numbers of white daughters and
orange-red sons. Crossovers or genetic imbalances occur at a rate of about 0.005–0.5%,
making coloration a reliable marker of genetic
sex (Hishida 1965).
In addition to the genetic marker, there is
distinct sexual dimorphism in medaka. Males
bear a notched dorsal fin, an anal fin with a
convex and serrated posterior margin, and an
elongate penultimate anal fin ray that give
their anal fins a square appearance. In addition, papillar processes form on the rays of
the anal fin in breeding males. Females lack
the dorsal notch, and their anal fins are more
concave, smooth at the margin, and lack
extended rays.
Injection exposure. Preparation. Details for
embryo exposure and rearing are provided in
Papoulias et al. (1999). Embryos were collected
Address correspondence to D.M. Papoulias, U.S.
Geological Survey, Columbia Environmental
Research Center, 4200 New Haven Road, Columbia,
MO 65201 USA. Telephone: (573) 876-1902. Fax:
(573) 876-1896. E-mail: Diana_Papoulias@usgs.gov
We thank E. Nelson, A. Allert, R. Claunch, L.
Patton, and E. Greer.
Funding and support were provided by the
Biological Resources Division, U.S. Geological Survey;
the Missouri Cooperative Fish and Wildlife Research
Unit (Missouri Department of Conservation,
University of Missouri, U.S. Geological Survey, and
the Wildlife Management Institute cooperating); and
the E.I. Dupont de Nemours Company.
Received 12 February 2002; accepted 24 May 2002.
29
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Papoulias et al.
in the morning of the day before exposure and
embedded in a solid 1% agarose (Sigma
Chemical Co., St. Louis, MO) matrix that had
been set up in square 36-grid petri dishes
(Falcon, Lincoln Park, NJ). The agarose
matrix provided the necessary stability for
injecting the embryos. The dish was then filled
with well water and stored in a cool place
(~20°C) until injection (within 24 hr).
Injection. Immediately preceding injection, dead embryos or embryos that had completed epiboly were replaced, and the well
water covering the embryos was replaced with
a sterile saline solution. Aluminosilicate capillary tubes (1.0 mm outer diameter and 0.53
internal diameter; Sutter Instrument Co.,
Novato, CA) were used to make injection needles with 5–10 µm internal-diameter tips. A
calculated volume of material (~0.1% of egg
volume; egg weight, 1.0 mg ± 0.2 mg/egg)
was injected directly into the oil globule using
a Narashigi picoinjector (PLI-188; Nikon,
Inc., Melville, NY) and micromanipulator
(MM-3; Stoelting Co., Wood Dale, IL). The
injected embryos were left in the agarose at
room temperature until the next day.
Incubation. Approximately 24 hr after
injection, dead embryos were culled and
recorded, and the survivors were placed in well
water in 150-mL glass side-arm test tubes
(Corning, Corning, NY) for incubation (rolled
at 27°C by a gentle stream of air). Dead
embryos were removed daily and counted.
Grow-out. Upon hatch, the medaka larvae were moved to 12.7 × 12.7 × 16.5 cm
containers suspended in 137.2 × 35.6 × 30.5
cm stainless steel raceways supplied with a
constant flow of well water (~300 mL/min).
An 18:6 hr light:dark photoperiod and
25–27°C temperatures were maintained
throughout rearing. Three times per day, the
newly hatched fry were fed day-old brine
shrimp (Artemia salina) and finely ground
flake food (Wardley Spectra IV; Secaucus,
NJ). After reaching 2 weeks of age, the
medaka were provided 2-day-old brine
shrimp and flake food. Feeding continued at
three times daily during the week and once
per day on weekends. The fish were moved to
12.7 × 15.2 × 22.9 cm stainless steel screen
cages (200 mm2 mesh) after 1–2 months and
were maintained there until they reached
maturity. Research was conducted in accordance with the CERC Animal Welfare Plan
and approved by CERC’s Animal Care and
Use Committee.
Experimental design. o,p´-DDE (99.8%
pure; Chem Service, Westchester, PA) was
dissolved in methylene chloride to the appropriate concentrations via serial dilution. After
transfer to sterile triolein (95% pure, Sigma),
the carrier of choice for these injections, the
methylene chloride was removed by evaporation. Embryos (36 per treatment) were
injected with a 0.5 nL volume of triolein
alone (control) or one of three concentrations
of o,p´-DDE (0.5, 0.05, 0.005 ng/embryo) in
the carrier triolein. Adult fish were identified
as genetic males (XY, orange-red) or genetic
females (XX, white) at 107 days after fertilization and were measured (total length) and
weighed (blotted dry) to assess growth. All
were sacrificed in a concentrated solution of
MS-222 (Sigma), and the gonads were dissected from a subsample of five white and five
red fish to assess gonadosomatic index (GSI),
sex reversal, and gonad histopathology. GSI
was calculated as gonad weight divided by
somatic weight × 100. Sex reversal was determined by comparing fish color (genetic sex)
to the phenotypic sex as determined by
inspection of the gonad. Histologic analysis
of the preserved gonads (Bouin’s solution;
Sigma) followed standard procedures for processing, dewaxing, hydration, and dehydration (Luna 1968). All sections were cut at 7
µm and stained using hematoxylin and eosin.
Data analysis. Mean body weight, body
length, and GSI were tested for differences
among control and o,p´-DDE-treated groups.
Values for GSI ratios were arcsine transformed before analysis using one-way analysis
of variance (ANOVA), testing males and
females separately. We used a two-way
ANOVA to test the interaction of sex and
treatment on weight and length. A general
linear model (α = 0.05) and least-squares
means were used to detect any significant differences. The tests were performed using SAS
software (SAS 2000).
Results
Survival and growth. Survival was highest in
the triolein-exposed fish (69%) and lowest in
the 0.5 ng o,p´-DDE/embryo treatment
(44%). Fish from the highest o,p´-DDE
exposure (0.5 ng/embryo) were significantly
larger than fish exposed to lower doses or the
control (Table 1), and there was no significant interaction between treatment and color
(genetic sex) on fish size.
Gonadosomatic index. GSI was inversely
related to o,p´-DDE exposure for both males
and females (Table 1). The lower GSI values
for the exposed fish resulted from greatly
decreased gonad weights.
Secondary sexual characteristics. Anal fin
breeding tubercles or processes were observed
on all triolein-injected males but on only
one o,p´-DDE-injected individual (0.005
ng/embryo). No tubercles were observed on
any of the embryos injected with 0.05 or 0.5
ng o,p´-DDE. All of the other secondary sexual characteristics (i.e., fin morphology)
appeared appropriate for the sex phenotype.
Gonadal histopathology. Histologic examination of the gonads of the o,p´-DDE-exposed
males and females indicated that no sex reversal or intersexes were caused by o,p´-DDE.
However, female medaka exposed in ovo to
o,p´-DDE did display gonadal abnormalities.
All treated females possessed very few vitellogenic oocytes and exhibited an increased
incidence of atresia, whereas untreated female
gonads appeared mature, had empty follicles,
and contained oocytes at various developmental stages (Figure 1A and B). The testes
of both treated and untreated males were
mature, but testes were markedly smaller in
treated fish (Figure 1C and D).
Discussion
Male-to-female sex reversal was not observed
at any dose of o,p´-DDE (0.005–0.5 ng/egg)
in our study. This differs from the results of
Stewart et al. (2000), who did observe maleto-female sex reversal in d-rR medaka exposed
in ovo to o,p´-DDT. This inconsistency may
be due to the differences between the parent
compound and the metabolite or, more
likely, the concentration: in the experiments
Table 1. Total length, weight, and GSI [mean ± SD (n)] of adult males and females exposed to different doses of o,p´-DDE in ovo.
o,p´-DDE
(ng/embryo)
Length (mm)
Female
Male
0
26 ± 1
(16)
27 ± 2
(9)
26 ± 1
(12)
29 ± 1*
(9)
0.005
0.05
0.5
27 ± 2
(8)
27 ± 1
(12)
27 ± 2
(10)
29 ± 1**
(7)
Weight (g)
GSI
Female
Male
Female
Male
0.16 ± 0.02
(16)
0.17 ± 0.03
(9)
0.15 ± 0.02
(12)
0.21 ± 0.03#
(9)
0.18 ± 0.03
(8)
0.18 ± 0.02
(12)
0.17 ± 0.04
(10)
0.23 ± 0.04**
(7)
3.12 ± 2.11
(5)
1.42 ± 0.21##
(5)
ND
0.58 ± 0.24
(5)
0.09 ± 0.07##
(5)
0.09 ± 0.05##
(4)
0.09 ± 0.04##
(5)
0.87 ± 0.11##
(5)
Gonad weight
Female
Male
0.0048 ± 0.003
(5)
0.0026 ± 0.0006
(5)
ND
0.0019 ± 0.0002
(5)
0.001 ± 0.0006
(5)
0.0002 ± 0.0001
(5)
0.0002 ± 0.0001
(4)
0.0002 ± 0.0001
(5)
ND, no data available for gonad weights of females.
*Longer than triolein-injected controls and 0.05-exposed females (p < 0.05). **Longer or heavier than triolein-injected controls and lower dose exposed males (p < 0.05). # Heavier than triolein-injected controls and lower dose exposed females. ##Lower GSI than triolein-injected controls (p < 0.05).
30
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111 | NUMBER 1 | January 2003 • Environmental Health Perspectives
Articles
of Stewart et al. (2000), 227 ± 22 ng o,p´DDT/egg was required for sex reversal, an
exposure approximately 400 times our highest
concentration. Metcalfe et al. (2000) have
reported that maternal exposure to o,p´-DDT
(egg concentrations estimated at 80–102
ng/egg) failed to produce intersex progeny.
Although the lack of male offspring produced
by treated females might have been indicative
of complete feminization of genetic males,
these authors did not consider this finding
definitive evidence of sex reversal because their
experiments were not conducted with fish that
had a male genetic marker (e.g., d-rR strain).
Carlson et al. (2000) injected groups of
mixed-sex and monosex rainbow trout
(Oncorhynchus mykiss) and mixed-sex chinook
salmon (Oncorhynchus tshawytscha) embryos
with o,p´-DDE in ranges of 10–160 mg/kg
and 1–80 mg/kg, respectively. In their first set
of trout experiments, these authors observed
an elevated male:female sex ratio at 80 and
160 mg/kg and a single intersex individual at
the lethally toxic 160 mg/kg exposure. Their
subsequent experiments with trout and
salmon did not indicate an increase in males
over females, and no further evidence of intersex was found. The lack of an effect on sexual
phenotype after in ovo o,p´-DDE exposure in
the experiments of Carlson et al. (2000) are
consistent with our results with medaka.
•
o,p´-DDE affects medaka sexual development
However, whereas we observed a marked
effect on GSI, they reported none for the subsample of females from the mixed-sex trout
experiment that were reared to maturity; male
GSI was not reported. Carlson et al. (2000)
concluded that o,p´-DDE-induced mortality
was likely to mask the more subtle effects on
sexual development.
The observed effects on gonadal development and histopathology in the present study
are consistent with some previous studies of
effects of DDT exposure on fishes, yet inconsistent with other studies. Female white croakers (Genyonemus lineatus) collected from San
Pedro Bay, California, with body burdens of
≥ 3.8 ppm ΣDDT (total of all isomers of
DDT and its breakdown products) could not
be induced to spawn and exhibited only earlystage oocytes, of which high numbers were
atretic relative to reference fish (Hose et al.
1989). In contrast to our results, Metcalfe et
al. (2000) reported advanced oogenesis in
female medaka developed from eggs with
o,p´-DDT concentrations of approximately
80–102 ng/egg, although both reference and
treated groups began producing viable eggs at
the same time. Males in this same study did
not display small gonads and associated secondary sexual effects (lack of breeding tubercles) that we observed. However, when
Metcalfe et al. (2000) exposed these male
Figure 1. Gonadal sections of female (A and B) and male (C and D) of adult d-rR medaka exposed in ovo to
triolein solvent (A and C) and 0.5 ng o,p´-DDE/embryo (B and D).
Environmental Health Perspectives
• VOLUME 111 | NUMBER 1 | January 2003
progeny to estradiol at 10 months of age, they
found that the male offspring showed significantly greater induction of vitellogenin than
did controls, suggesting that early DDT
exposure may have potentiated vitellogenin
induction later in life.
The effects of o,p´-DDE we observed on
gonadal development in fish are also similar to
the effects reportedly caused by estrogen exposure. In ovo exposure of medaka to ethinyl
estradiol also produced fish with small ovaries
with mostly perinuclear and atretic oocytes,
and normal, but less mature, testes (Papoulias
et al. 1999). Direct comparisons between
results of other estrogen exposure studies and
our o,p´-DDE results may be questionable
because of differences in developmental stages
at which fish were exposed. Adult fathead
minnow (Pimephales promelas) males exposed
for 21 days to low concentrations of estrogen
and estrone showed complete inhibition of
testicular growth during recrudescence (Panter
et al. 1998). Exposing sexually mature male
and female fathead minnows to 17α-estradiol
for 14 days caused a reduction in male secondary sexual characteristic development and
degenerative testis changes, whereas ovarian
development appeared retarded and produced
more atretic oocytes than in unexposed
females (Miles-Richardson et al. 1999). It is
interesting to note that male summer flounder
(Paralichthys denatus) exposed as juveniles to
o,p´-DDT displayed degenerative features
similar to those in adult fatheads exposed to
estrogen (Zaroogian et al. 2001), perhaps further supporting the idea that exposure effects
will vary depending on the life stage at which
exposure occurs.
Estrogen exposure typically has the toxic
effect of repressing gonadal and somatic
growth; consequently, we do not consider that
the larger size of the fish in our highest o,p´DDE treatment was due to anabolic effects of
the xenoestrogen (Donaldson et al. 1979;
Papoulias et al. 1999). Although we attempted
to feed our fish to excess, we cannot be certain
that the body size differences we saw were due
to an o,p´-DDE effect because of the lowered
survival of the highest treatment group.
Higher mortality resulted in lower tank density, potentially allowing more food per fish in
these treatments. Nevertheless, gonad weight
relative to somatic weight decreased with
increasing o,p´-DDE concentration. We suggest that exposure to nonlethal concentrations
of (xeno)estrogens early in development may
be analogous to chemical castration in mammals, which diverts energetic resources normally used to develop mature gonads toward
somatic growth.
Our results indicate that male medaka
embryos exposed to ≤ 0.5 ng/embryo of
o,p´-DDE are not at risk of being feminized.
Although estrogens at this concentration can
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Papoulias et al.
cause sex reversal (Papoulias et al. 1999), the
comparatively lower affinity of o,p´-DDE and
o,p´-DDT for the ER (Nimrod and Benson
1997) suggests that higher exposures of these
compounds are required for sex reversal to
occur. Nevertheless, o,p´-DDT and o,p´-DDE
at low concentrations may interfere with the
binding of natural ligands to a variety of
steroid binding moieties (e.g., receptors and
binding proteins) (Danzo 1997; Donohoe
and Curtis 1996; Gaido et al. 1997; Kupfer
and Bulger 1976; Lundholm 1998; Mason
and Schulte 1980; Nimrod and Benson
1997; Shilling and Williams 2000; Wells and
Van Der Kraak 2000), thereby allowing for
endocrine-disrupting effects.
Whether the effects on gonad development that we observed in the medaka fish
result from disruption of the endocrine system specifically or are a consequence of other
mechanisms of toxicity was not determined
in the present study. Chemically induced
genotoxic and nongenotoxic effects during
development may manifest at early or later
ontogenetic stages (McNabb et al. 1999).
Although our method of exposing the developing embryos through injection mimics maternal transfer of contaminants, it also allows for
disruption of the development and differentiation of many tissues and processes specifically
occurring during embryogenesis. Reproductive
toxicity of the o,p´-DDE-exposed medaka in
our study was evident by the small, underdeveloped gonads and suggests early impairment
along the brain–pituitary–gonadal axis.
Few data are available regarding environmental concentrations of o,p´-DDE in eggs
and embryos from which risks to fish populations can be assessed. Concentrations found
in Lake Michigan salmonid eggs have been
reported to range from 3 to 150 µg/kg (Giesy
et al. 1986; Miller 1993). Data collected in
the early 1990s from the northwestern
United States indicate that fish tissue concentrations of 8–22 µg/kg may still be encountered (Brown 1997; Munn and Gruber
1997). At these o,p´-DDE concentrations,
toxic effects were observed in our medaka.
We have shown that the reproductive
development of both males and females may
be impaired by maternally derived concentrations of o,p´-DDE as low as 0.005 ng/embryo
(or 5 ng/g egg). Our observations of small but
normal male testes and abnormal female
ovaries after in ovo o,p´-DDE exposure may
be indicative of disruption of early endocrine
32
processes that lead to normal ovarian and testicular development. Although o,p´-DDE
body burdens are generally quite low (parts
per billion), our data suggest that o,p´-DDE
may affect sexual development but is unlikely
to affect sexual differentiation in offspring of
most exposed fishes.
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111 | NUMBER 1 | January 2003 • Environmental Health Perspectives